Depressed neuromuscular transmission causes weakness in mice lacking BK potassium channels

Abstract

Mice lacking functional large-conductance voltage- and Ca2+-activated K+ channels (BK channels) are viable but have motor deficits including ataxia and weakness. The cause of weakness is unknown. In this study, we discovered, in vivo, that skeletal muscle in mice lacking BK channels (BK-/-) was weak in response to nerve stimulation but not to direct muscle stimulation, suggesting a failure of neuromuscular transmission. Voltage-clamp studies of the BK-/- neuromuscular junction (NMJ) revealed a reduction in evoked endplate current amplitude and the frequency of spontaneous vesicle release compared with WT littermates. Responses to 50-Hz stimulation indicated a reduced probability of vesicle release in BK-/- mice, suggestive of lower presynaptic Ca2+ entry. Pharmacological block of BK channels in WT NMJs did not affect NMJ function, surprisingly suggesting that the reduced vesicle release in BK-/- NMJs was not due to loss of BK channel-mediated K+ current. Possible explanations for our data include an effect of BK channels on development of the NMJ, a role for BK channels in regulating presynaptic Ca2+ current or the effectiveness of Ca2+ in triggering release. Consistent with reduced Ca2+ entry or effectiveness of Ca2+ in triggering release, use of 3,4-diaminopyridine to widen action potentials normalized evoked release in BK-/- mice to WT levels. Intraperitoneal application of 3,4-diaminopyridine fully restored in vivo nerve-stimulated muscle force in BK-/- mice. Our work demonstrates that mice lacking BK channels have weakness due to a defect in vesicle release at the NMJ.

Figure 1.

Weakness in BK^−/− mice is due to neuromuscular dysfunction. (A) Representative specific force traces from WT (black) and BK^−/− (red) muscle showing twitches (top panel) and responses to 30-Hz (middle panel) and 60-Hz (bottom panel) stimulation for both nerve stimulation (left column) and direct muscle stimulation (right column). (B) Bar graph showing twitch-specific force for nerve and muscle stimulation. Values shown as mean ± SEM. *, P < 0.05, one-way ANOVA. (C) Specific force–frequency curves, fitted with a Boltzmann equation, for WT nerve (black solid line) and muscle (black dashed line) stimulation as well as BK^−/− nerve (red solid line) and muscle (red dashed line) stimulation. Values are shown as ± SEM. n = four WT and six BK^−/− mice.

Figure 2.

Vesicle release is reduced at the NMJ of BK^−/− mice. (A) Representative average traces of the evoked EPC and the spontaneous mEPC from WT (black traces) and BK^−/− (red traces) NMJs. (B) Plotted mean values for measures of synaptic function for WT (black) and BK^−/− (red) NMJs. EPC and mEPC peak amplitudes were measured in nanoamps (nA); Quantal content is the number of vesicles released per presynaptic action potential (EPC peak amplitude/mEPC peak amplitude). mFreq, frequency of mEPCs in the absence of nerve stimulation. Values are shown as ± SEM. **, P < 0.01. n = 111 NMJs from nine WT mice and 98 NMJs from eight BK^−/− mice.

Figure 3.

BK^−/− NMJs exhibit facilitation rather than depression during repetitive stimulation. (A) 10 EPCs during a 50-Hz train of pulses for a representative WT (black) and BK^−/− (red) NMJ. In both traces, stimulus artifacts were removed for clarity. (B) The change in average EPC amplitudes during the 50-Hz trains for WT and BK^−/− NMJs. WT NMJs showed a slight increase in EPC amplitude early in the train of stimuli (facilitation); at the end of the train, EPC amplitude decreased below the initial value (depression). In BK^−/− NMJs, facilitation was maintained throughout the train of stimuli. (C) Facilitation in BK^−/− NMJs was significantly increased for both the second (P2/P1) and tenth stimuli (P10/P1) of the train (**, P < 0.01 versus WT for both comparisons). Values are shown as ± SEM. n = 93 NMJs from nine WT mice and 83 NMJs from eight BK^−/− mice.

Figure 4.

The reduction in vesicle release at BK^−/− NMJs can be overcome by blocking voltage-gated K⁺ channels. (A) Superimposed representative WT (black) and BK^−/− (red) EPCs and mEPCs before (solid lines) and after (dashed lines) treatment with 100 µM 3,4-DAP. (B) Bar graphs show mean data ± SEM. n = five WT and five BK^−/− mice. At least seven NMJs were recorded from each muscle. *, P < 0.05; **, P < 0.01.

Figure 5.

Normalization of BK^−/− muscle force production following treatment with 3,4-DAP. (A and B) Representative twitch (A) and tetanic (60 Hz; B) muscle force traces following stimulation of the sciatic nerve of WT without (black solid line) and with 3,4-DAP (black dashed line) as well as BK^−/− without (red solid line) and with 3,4-DAP (red dashed line). (C) Specific force–frequency curves, fitted with a Boltzmann equation. Values are mean ± SEM. n = five WT and four BK^−/− mice.